Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A

A marine cyanobacterial cyclic depsipeptide, coibamide A (CbA), inhibits the mammalian protein secretory pathway by blocking the Sec61 translocon, which is an emerging drug target for cancer and other chronic diseases. In our previous structure–activity relationship study of CbA, the macrolactone ester linker was replaced with alkyl/alkenyl surrogates to provide synthetically accessible macrocyclic scaffolds. To optimize the cellular bioactivity profile of CbA analogues, novel lysine mimetics having β- and ε-methyl groups have now been designed and synthesized by a stereoselective route. A significant increase in cytotoxicity was observed upon introduction of these two methyl groups, corresponding to the d-MeAla α-methyl and MeThr β-methyl of CbA. All synthetic products retained the ability to inhibit secretion of a model Sec61 substrate. Tandem evaluation of secretory function inhibition in living cells and cytotoxicity was an effective strategy to assess the impact of structural modifications to the linker for ring closure.

C oibamide A (CbA, 1a) is a cyclic depsipeptide isolated from a Panamanian marine filamentous cyanobacterium. 1 The highly N-methylated peptide, with one D-amino acid and a D-hydroxy acid (Figure 1A), exhibited potent antiproliferative activity against several human cancer cell lines. 1,2Using a synthetic CbA photoaffinity probe, we previously revealed that CbA binds to the α subunit of the Sec61 translocon (Sec61α) and prevents insertion of secreted and membrane proteins into the endoplasmic reticulum (ER) membrane or across the ER membrane into the ER luminal space. 3To date, several studies have examined the biological activity of CbA and the downstream target(s) that are impacted by the CbA-induced block of Sec61-dependent protein biosynthesis in the ER secretory pathway.First discovered in a screen for anticancer activity, 1 CbA induces cell-cycle arrest, inhibits cancer cell migration and invasion in vitro, and retains antitumor properties in subcutaneous xenograft mouse models. 4This bioactivity profile may be mediated, at least in part, by inhibition of extracellular secreted proteins, such as vascular endothelial growth factor A (VEGF-A), as well as by decreases in the expression of integral membrane receptors, such as vascular endothelial growth factor receptor 2 (VEGFR2), and human epidermal growth factor receptor (HER) members, especially EGFR (ErbB-1) and HER3 (ErbB-3). 4,5CbA also induces macroautophagy via a mammalian target of rapamycin (mTOR)-independent mechanism. 6CbA-induced autophagy, following short-term exposure of cells, depends on the presence of autophagy-related protein 5 (ATG5), and eventually defects in the autophagosome−lysosomal fusion promote regulated cell death signaling. 7,8here are a number of other natural products and synthetic molecules that selectively inhibit protein secretion via Sec61α inhibition, including apratoxin A, 9,10 decatransin, 11 eeyarestatin I, 12,13 HUN-7293, 14−16 ipomoeassin F, 17 and mycolactones A/ B 18 (Figure S3).Among these compounds, synthetic cotransins prevent Sec61-mediated protein biosynthesis in a relatively substrate-specific manner, while cytotoxic natural products such as apratoxin A, ipomoeassin F, and coibamide A (CbA) inhibit co-translational translocation of a broad range of Sec61 substrates with variable sensitivity. 19Interestingly, although these natural products all bind to Sec61α, when screened against the National Cancer Institute 60 (NCI 60) human tumor cell line panel, individual Sec61 inhibitors had distinct profiles. 3These findings suggest that the natural products may bind to Sec61α in different ways and/or some cancer cells are differentially sensitive to natural Sec61 inhibitors, resulting in a wide range of cellular responses.Alterations of the CbA structure may improve the substrate specificity of Sec61α inhibition as well as the target selectivity, leading to development of potential drug candidates with favorable biological and physicochemical profiles.Thus, we investigated the structure−activity relationships (SARs) of CbA and derivatives in order to characterize key structural elements required for indispensable binding interactions as well as for attractive bioactivities.
Yao et al. reported an SAR study which demonstrated that the potent cytotoxic activity of CbA was maintained when the two N,O-dimethylserines [MeSer(Me)] in CbA were replaced with N-methylalanines (MeAla), whereas most derivatives with simplified structures were less potent (Figure 1A). 20We also previously investigated modification of the CbA macrolactone substructure. 21CbA mimetic 2a, in which the D-MeAla 11 -MeThr 5 moiety at the ring junction was substituted with a simple MeLys(Me) (Figure 1B), displayed moderate cytotoxic activity. 21This lysine-linked CbA mimetic was applicable to our lead optimization campaign, in which we revealed that substitution of Tyr(Me) 10 in CbA with (biphenylyl)alanine (Bph) led to a significant increase in cytotoxic potential.The modifications of CbA with MeLys(Me) facilitated the synthesis of a series of derivatives to avoid possible racemization and less efficient couplings.However, the cytotoxic potential of 2a was significantly less than CbA.These data suggested that the MeThr 5 β-methyl and D-MeAla 11 α-methyl in CbA would contribute to the cellular bioactivity via the direct interaction with the target molecule(s) and/or other indirect effect(s).The presence of these backbone methyl groups and their configurations alter the local conformations by steric hindrance and may consequently modify the patterns of intramolecular hydrogen bonding to affect the global conformations of peptide macrocycles. 22These may further optimize the interaction of the side chain(s) with the target molecule and/or increase the cell membrane permeability.We envisioned that the introduction of two methyl groups in the corresponding positions and stereochemical configurations would improve the cellular bioactivity of CbA mimetics.Initially, we designed two cyclic peptides 3a,b incorporating novel lysine derivatives at the ring junction, in which two methyl groups corresponding to the MeThr 5 β-methyl and D-MeAla 11 α-methyl in CbA are positioned at the lysine βand εpositions (Figure 1C).
For the preparation of peptides 3a,b by a standard Fmocbased solid-phase peptide synthesis (SPPS), we designed two novel lysine derivatives, 4a,b (Figure 1D).In order to distinguish two amino groups in the lysine analogues for the site-selective acylation, Fmoc and Alloc groups were employed for the αand ε-amino groups, respectively.The three configurations of the α-amino, β-methyl, and ε-methyl groups should be selectively constructed for the synthesis of 4a,b.Concomitant control of the E-alkene geometry existing between two asymmetric carbons in 4b is also needed.
Synthesis of the two lysine analogues began with D-serine- derived Garner aldehyde (Scheme 1). 23Initially, Garner aldehyde 5 was subjected to a Wittig reaction with (triphenylphosphoranylidene)acetaldehyde to give α,β-unsaturated aldehyde 6. 24 n-Bu 2 BOTf-mediated Evans aldol reaction of 6 provided the syn-aldol product 7 having an α-methyl group which becomes the ε-methyl in 4a,b. 25,26The Sconfiguration of the hydroxy group in 7 is important for the late-stage stereoselective introduction of the β-methyl group in 4a,b.Protection of the hydroxy group in 7 followed by hydrolytic removal of the chiral auxiliary afforded acid 9.The stereochemistry of 9 was confirmed by X-ray crystallography (Figure S1).Diphenylphosphoryl azide (DPPA)-mediated conversion of the acid 9 into acyl azide followed by Curtius rearrangement provided an isocyanate intermediate, which was treated with benzyl and allyl alcohols to give Cbz-and Allocprotected amines 10a,b, respectively.In this step, the Cbz group was employed as an interim protecting group for the synthesis of 10a to avoid the loss of function of the Alloc group by the later olefinic hydrogenation.After deprotection of the TBS group, the resulting hydroxy group was activated as mesylates 12a,b.Introduction by site-and stereoselective anti-S N 2′ alkylation of 12a,b was achieved using organocopper reagents to provide β-methyl adducts 13a,b. 27,28The configuration at the β-position in 13b was confirmed by comparative NMR analysis with an authentic sample, which was synthesized via an alternative route (Scheme S1).Olefin hydrogenation in 13a and simultaneous Cbz deprotection followed by Alloc protection gave lysine precursor 14 with an aminoalkyl side chain.Protecting group manipulations in acetonides 13b and 14 followed by AZADOL-mediated oxidation of the resulting alcohols 15a,b afforded the expected lysine derivatives 4a,b.
Next, we synthesized CbA derivatives 3a,b by Fmoc-based SPPS using Fmoc/Alloc protected lysine derivatives 4a,b, according to the synthetic procedure for CbA mimetics in our previous study (Scheme S2). 21The bioactivity profile of the resulting peptides 3a,b was investigated by using a standard MTS antiproliferative/cell viability assay to assess the cytotoxic potential of CbA analogues against human A549 lung cancer cells (Table 1).Peptide 3a, with a 3,6-dimethyllysine moiety at the ring junction, showed enhanced cytotoxic activity compared to that of nonmethylated peptide 2a [CC 50 (3a) = 41 nM].Peptide 3b, with restricted rotation of the C γ �C δ double bond in the lysine linker, was equipotent with the parent ester 1b [CC 50 (3b) = 2.6 nM], and also 10-fold more potent than 3a.As such, the two βand ε-methyls in the lysine linkages of 3a,b, which correspond to the MeThr 5 β-methyl and D-MeAla 11 α-methyl in CbA, contribute to potent bioactivity, possibly due to direct interactions with the target molecule and/or an indirect effect on the bioactive conformations.Additionally, the presence of a δ-C(sp 2 ) in the lysine linker of 3b and the corresponding D-MeAla 11 carbonyl carbon in 1b would contribute to stabilization of favorable CbA macrocycle conformations.
Given the favorable effect of limited ring junction flexibility on bioactive conformations of the macrocycle, caused by the ester bond in 1a,b and olefin in 3b, we subsequently investigated the SARs of amide moieties at the ring junction.Amides and alkenes have been used as mutually exchangeable isosteres in medicinal chemistry 28−32 because their planar architecture maintains the parent structure to reproduce desired bioactivities. 33−37 In order to investigate the effect on bioactivity of a macrolactam, we designed and synthesized peptide 16a, in which an amide bond was substituted for the D-MeAla 11   Thus, it is inferred that substitution of the CbA ester bond with the amide bond in 16a−c does not alter the conformation of these bioactive peptides significantly.The amide NH hydrogen bond donor in 16a−c did not lead to unfavorable interactions or, presumably, a significant change in macrocyclic conformation.In the case of CbA amide analogues, the presence of the β-methyl group in Dab 5 , which corresponds to the MeThr 5 β-methyl in CbA, was indispensable for potent cytotoxic activity.
Representative CbA peptides from the SAR study were evaluated for cytotoxic activity against two additional human cancer cell lines and normal human dermal fibroblasts (HDFs) (Table S1).Consistent with our previous studies, CbA (1a) showed low nanomolar toxicity to HCT116 colon cancer cells and was approximately 30-fold less potent against U87-MG glioblastoma cells. 3,6The SAR for cytotoxic potential of the CbA derivatives against HCT116 colon, U87-MG glioblastoma, and normal HDFs was roughly the same as that observed for A549 lung cancer cells employed for the initial SAR analysis, although cell-type specific differences in sensitivity were also observed (Table S1).Among the CbA analogues investigated, Bph 10 analogue 16c was the most potent cytotoxic peptide against all three cancer cell lines and HDFs.Peptides 2a and 17a with no β-methyl groups at position 5 exhibited less cytotoxicity against all cell lines compared with other peptides possessing two methyl groups at positions corresponding to those of the original D-MeAla 11 -MeThr 5 .Peptides 2a, 3a, and 17a were generally least toxic to normal HDFs although these cells also displayed a distinct pattern of sensitivity (Table S1).Taken together, these data suggest that the appropriate arrangement of the methyl groups around the ring junction plays an important role in cellular bioactivity and that the relative safety of individual peptides against normal cells cannot necessarily be predicted a priori due to heterogeneity in the sensitivity of various histological cancer cell types to CbA 1 and CbA analogues.
To investigate the mechanistic basis for cytotoxicity, all CbA derivatives were evaluated for their ability to inhibit expression of a model Sec61 substrate using U87-MG cells engineered to express a naturally secreted reporter protein, Gaussia luciferase (GLuc) (Tables 1 and 2).For these studies, U87-MG-GLuc cells were exposed to increasing concentrations of each peptide, or vehicle, for 18 h, and the cell culture medium was assayed for the presence of GLuc.All CbA derivatives showed concentration dependent inhibition of GLuc secretion (Figure S4).Both CbA (1a) and MeAla 3 /MeAla 6 analogue 1b showed potent inhibition of GLuc secretion.The methyldeficient analogue 2a, and modification of the D-MeAla 11 -MeThr 5 substructure in CbA with lysine derivatives in peptides Cytotoxic concentration (CC 50 ) values are the concentrations needed for 50% inhibition of human A549 lung cancer cells relative to control (n = 3).b Inhibitory concentration (IC 50 ) values are the concentrations needed for 50% inhibition of GLuc expression.The secretory function of U87-GLuc cells was measured after 18-h incubation in the presence or absence of the compound (n = 3).At the 18-h end point of the protein secretion assay in U87-GLuc cells, no effect on cell viability was observed at 0.03 to 300 nM for CbA (1a) and 0.3 nM to 3.0 μM for all other peptides.c Not tested.
3a,b, resulted in a slight decrease in potency.Interestingly, peptides 16a−c with an amide surrogate at the ring closure exhibited potent (<1 nM) inhibition of GLuc protein secretion.In particular, peptides 16b and 16c which retain the MeSer(Me) 3 /MeSer(Me) 6 in CbA exhibited more than a 10-fold increase in potency of GLuc inhibition relative to CbA (1a).Notably, the methyl-deficient derivatives 2a and 17a were less potent inhibitors of GLuc secretion, suggesting that the presence of two methyl groups corresponding to the MeThr 5 β-methyl and D-MeAla 11 α-methyl in CbA contribute to functional inhibition of GLuc secretion.Independent evaluation of the ability of peptides 2a, 3a, and 16b to inhibit expression of secreted VEGF-A from human SF-296 glioblastoma cells was also consistent with the pattern of activity observed for GLuc inhibition (Figure S5).All peptides induced statistically significant and concentration-dependent decreases in expression of endogenous VEGF-A; however, a ranked order of decreasing potency was observed as follows: 16b ≤ 1a < 3a < 2a (Figure S5).
These investigations revealed that there are differences in cytotoxic potential of CbA analogues harboring modifications at ring closure.Further, all CbA analogues inhibited secretion of a GLuc reporter protein in living cells, providing independent confirmation of Sec61 target engagement and additional refinement of the SAR established through basic cell viability assays.We can assume that the enhanced cellular bioactivity of macrolactam derivatives 16b,c, relative to 1a, are derived predominantly from potent inhibition of Sec61dependent secretory function, resulting in broad inhibition of Sec61 substrate biosynthesis and enhanced cytotoxicity. 38In contrast, lysine derivatives 3a,b were over 10-fold less potent inhibitors of GLuc secretion than 1a,b, and retained the same rank order for inhibition of GLuc secretion and general cytotoxic potency with peptide 3a showing a better safety profile against normal HDFs than 3b (Table S1).However, the ability of 2a to retain GLuc inhibitory potency (where 2a ≈ 3a), was not predicted by assessment of cell viability alone (Tables 1 and S1) Thus, peptide 2a represents an example of the potential to separate functional inhibition of secretory pathway function from overt cytotoxicity by chemical modification.The nonmethylated peptide 2a inhibited GLuc and VEGF-A secretion at nM concentrations (Figures S4 and  S5) yet was 160-fold less toxic to normal HDFs than 1a (Table S1).
In conclusion, we conducted an SAR study of the D- MeAla 11 -MeThr 5 substructure in CbA in order to establish alternative macrocycle linkers.Substitution of the ester linkage with an olefin or amide bond reproduced the potent cytotoxicity of CbA.The two methyl groups in this macrocycle linker, which correspond to MeThr 5 β-methyl and D-MeAla 11 α-methyl, are critical determinants of CbA bioactivity.Using a sensitive screening assay that reports on cellular secretory function, we established that modification of the ring junction with lysine mimetics preserves the ability of the molecule to inhibit GLuc secretion while inducing less overt cytotoxicity.In contrast, the macrolactam increased both GLuc inhibitory potency and cytotoxic activity relative to the natural product.The ability to interrogate SARs based on functional inhibition of secretion in living cells, in addition to the cytotoxic potential of CbA derivatives, indicates that the appropriate optimization of the macrocyclic scaffold of CbA through chemical synthesis could eventually lead to the development of more selective, less cytotoxic protein secretion inhibitors.Further SAR investigations as well as the investigations of the binding mode of CbA analogues are ongoing.
Experimental procedures of synthesis and biological evaluations, characterization data, and supporting figures (PDF)

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Scheme 1. Stereoselective Synthesis of Precursor Lys Derivatives a

Table 2 .
Structure−Activity Relationships of CbA Analogues with an Amide Linkage a